Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography.
Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.
Roos
P H
PH
Institut für Physiologische Chemie I. Abteilung Bioenergetik, Ruhr-Universität Bochum, F.R.G.
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification*; D008670:Metals; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D007527:Isoenzymes; D008670:Metals; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital